1. Field of the Invention
The invention relates to a process for the electrochemical regeneration of pyridine cofactors which are used in processes for the enzymatic synthesis of organic products.
2. Discussion of the Background
The study of the use of oxidation/reduction enzymes in organic synthesis is expanding rapidly. These enzymatic synthesis processes employ pyridine cofactors such as NADH (nicotinamide adenine dinucleotide) which are involved in the oxidation/reduction mechanism. However, extrapolation of the results obtained in the laboratory onto a larger scale necessitates regeneration of the pyridine cofactors, which are much too expensive to be used in stoichiometric amounts. It appears, moreover, that the absence of sufficiently efficient methods is preventing the development on an industrial scale of processes for preparing commercial products. For this reason, many studies are currently in progress aimed at finding the optimal conditions for regeneration of these cofactors. The regeneration may be carried out chemically, enzymatically or electrochemically. Analysis of the advantages and drawbacks of these various approaches shows that no general regeneration technique is completely satisfactory, and it is advisable to envisage, in each particular case, optimization of the overall direct reaction/regeneration system (Applied Biochemistry and Biotechnology, Vol. 14, 1987 pp. 147-197).
The methods of regeneration of NADH, that is to say reduction of the NAD.sup.+ form resulting from processes of reduction of the substrate to be converted, are illustrated in FIG. 1, in which:
S denotes the substrate to be converted. PA1 P denotes the synthesis product to be obtained. PA1 E.sub.1 E.sub.2 denote the enzymes involved in the mechanism, A and B the substrates and by products involved in the regeneration.
The mechanism a illustrates a chemical regeneration; the molecule bringing about the regeneration reduces NAD.sup.+ directly to NADH. Hydrogen, used under pressure, has been proposed as a reducing molecule (Biotechnology and Bioengineering, Vol. 7, No. 9, 1985, pp. 1277-1281), but its use gives rise to problems of implementation and safety.
The mechanism b illustrates an enzymatic regeneration with an enzyme which accepts several substrates: the same enzyme catalyses the synthesis and regeneration reactions.
The mechanism c illustrates a process for synthesis and regeneration each employing a different enzyme and substrate.
The mechanism d illustrates an electrochemical regeneration.
In the case of the reduction of NAD.sup.+, enzymatic methods have given the best results. Among enzymatic systems used, the following substrate/enzyme systems may be mentioned: formate/formate dehydrogenase, glucose 6-phosphate/glucose-6-phosphate dehydrogenase, glucose/glucose dehydrogenase, ethanol/alcohol dehydrogenase, hydrogen/hydrogenase.
The use has been proposed (Biotechnology letters 1983, 5(7), 463-468) of enzymes such as Alcaligenes eutrophus hydrogenase for reducing NAD.sup.+ to NADH with hydrogen. However, the stability of the bound enzyme was very low, in particular as a result of oxygen or various oxidizing agents, prohibiting the development of an efficient process which, moreover, in no instance envisages an implementation of the electrochemical type.
Electrochemical processes appear attractive, at least theoretically, since they make it possible to set the rate of regeneration very readily by the choice of electrode potential, and to avoid the use of the regeneration enzyme and reagent (FIG. 1). Moreover, they offer the possibility of a ready monitoring of the reaction by measuring the intensity of electrolysis during the process. However, the advantages are limited by the incompatibility of some reagents capable of reacting directly with the electrode brought to the reduction potential; poisoning of the electrode by adsorbable products and reactants and a lack of selectivity are other major drawbacks. The latter problem is particularly appreciable in relation to reduction, as a result o the formation by one-electron transfer of the free-radical intermediate NAD.sup.. which is capable of dimerizing rapidly. The radical appears on the electrodes irrespective of their nature.
Some efforts have been made to overcome this problem by modifying the surfaces by bound chemical mediators, as well as by the use of mediators in solution. These attempts have not yet enabled sufficient selectivity to be produce and, as a result, the direct or indirect electrochemical reduction of NAD.sup.+ has not been developed.
The electrochemical reduction of NAD.sup.+ has been envisaged most especially on a mercury electrode, on which there appears chiefly the dimer (NAD).sub.2, and NADH in a few special cases. The emphasis is placed most especially on the mechanistic aspect and on the adsorption phenomena.
On a bare platinum electrode, reduction of NAD.sup.+ gives a mixture of NADH and (NAD).sub.2 ; it takes place in the region of potential where gaseous hydrogen is evolved. The reduction is strongly dependent on the surface state of the electrode.
It has also been proposed (Journal of Biotechnology, vol. 1, 1984, pp. 95-109) to use mediators such as methyl viologen, often in combination with whole cells or cell extracts. In the mechanism, the mediator acts as a relay link in the transfer of electrons between the electrode and NAD.sup.+, and participates in a reaction catalyzed by the biosystem. It should be noted that the appearance of free-radical intermediates impairs the yield; in addition, the mediator/biosystem biocompatibility is reduced.